Method of using a small volume in vitro analyte sensor

Abstract

A sensor designed to determine the amount and concentration of analyte in a sample having a volume of less than about 1 μL. The sensor has a working electrode coated with a non-leachable redox mediator. The redox mediator acts as an electron transfer agent between the analyte and the electrode. In addition, a second electron transfer agent, such as an enzyme, can be added to facilitate the electrooxidation or electroreduction of the analyte. The redox mediator is typically a redox compound bound to a polymer. The preferred redox mediators are air-oxidizable. The amount of analyte can be determined by coulometry. One particular coulometric technique includes the measurement of the current between the working electrode and a counter or reference electrode at two or more times. The charge passed by this current to or from the analyte is correlated with the amount of analyte in the sample. Other electrochemical detection methods, such as amperometric, voltammetric, and potentiometric techniques, can also be used. The invention can be used to determine the concentration of a biomolecule, such as glucose or lactate, in a biological fluid, such as blood or serum. An enzyme capable of catalyzing the electrooxidation or electroreduction of the biomolecule is provided as a second electron transfer agent.

We claim: 
     
       1. A method of determining a concentration of an analyte in a sample, the method comprising steps of: contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, and a measurement zone bounded on at least two sides by the working electrode and counter electrode, wherein the measurement zone is sized to contain less than about 1 μL of sample;   holding the sample within the measurement zone in a non-flowing manner;   applying a potential between the working and counter electrodes to electrolyze a portion of the analyte in the sample in the measurement zone;   measuring current generated by the electrochemical sensor at a plurality of times while portion of the analyte is being electrolyzed; and   determining, by coulometry, a concentration of the analyte in the sample using the measured currents.   
     
     
       2. The method of claim 1, wherein the step of determining, by coulometry, a concentration of the analyte comprises extrapolating a current curve based on the measured currents. 
     
     
       3. The method of claim 2, wherein the step of determining, by coulometry, a concentration of the analyte further comprises determining, from the current curve, a charge necessary to electrolyze at least 90% of the analyte in the sample in the measurement zone, and   correlating the charge with the concentration of the analyte in the sample.   
     
     
       4. The method of claim 3, wherein the step of determining, from the current curve, a charge necessary to electrolyze at least 90% of the analyte in the sample in the measurement zone comprises integrating the current curve to determine the charge. 
     
     
       5. The method of claim 1, wherein the step of contacting the sample with an electrochemical sensor comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, and a measurement zone bounded on at least two sides by the working electrode and counter electrode, wherein the measurement zone is sized to contain less than about 0.5 μL of sample. 
     
     
       6. The method of claim 1, wherein the step of contacting the sample with an electrochemical sensor comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, and a measurement zone bounded on at least two sides by the working electrode and counter electrode, wherein the measurement zone is sized to contain less than about 0.2 μL of sample. 
     
     
       7. The method of claim 1, wherein the step of contacting the sample with an electrochemical sensor comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, a measurement zone bounded on at least two sides by the working electrode and counter electrode, and redox mediator disposed in the measurement zone, wherein the measurement zone is sized to contain less than about 1 μL of sample. 
     
     
       8. The method of claim 1, wherein the step of contacting the sample with an electrochemical sensor comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, a measurement zone bounded on at least two sides by the working electrode and counter electrode, and an electron transfer agent disposed in the measurement zone, wherein the measurement zone is sized to contain less than about 1 μL of sample. 
     
     
       9. The method of claim 1, wherein the step of contacting the sample with an electrochemical sensor comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode facing the working electrode, and a measurement zone between the working electrode and counter electrode, wherein the measurement zone is sized to contain less than about 1 μL of sample. 
     
     
       10. The method of claim 1, wherein the step of determining a concentration of the analyte in the sample comprises determining, by coulometry, a concentration of glucose in the sample using the measured currents. 
     
     
       11. The method of claim 1, wherein the step of determining a concentration of the analyte in the sample comprises determining, by coulometry, a concentration of glucose in a sample of blood using the measured currents. 
     
     
       12. The method of claim 1, wherein the step of contacting the sample with an electrochemical sensor comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode facing the working electrode and spaced apart by 0.2 mm or less, and a measurement zone between the working electrode and counter electrode, wherein the measurement zone is sized to contain less than about 1 μL of sample. 
     
     
       13. The method of claim 1, wherein the step of contacting the sample with an electrochemical sensor comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode facing the working electrode and spaced apart by 0.1 mm or less, and a measurement zone between the working electrode and counter electrode, wherein the measurement zone is sized to contain less than about 1 μL of sample. 
     
     
       14. The method of claim 1, wherein the step of applying a potential comprises electrolyzing the analyte in the sample in the measurement zone for a period of no more than one minute. 
     
     
       15. A method of determining a concentration of an analyte in a sample, the method comprising steps of: contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, and a sample chamber for holding the sample in electrolytic contact with the working electrode and counter electrode, wherein the sample chamber is sized to contain less than about 1 μL of sample;   holding the sample within the sample chamber in a non-flowing manner;   applying a potential between the working and counter electrodes to electrolyze a portion of the analyte in the sample within the sample chamber;   measuring current generated by the working electrode at a plurality of times while the portion of the analyte is being electrolyzed; and   determining, by coulometry, a concentration of the analyte in the sample using the measured currents.   
     
     
       16. The method of claim 15, wherein the step of contacting the sample with an electrochemical sensor comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, and a sample chamber for holding the sample in electrolytic contact with the working electrode and counter electrode, wherein the sample chamber is sized to contain less than about 0.5 μL of sample. 
     
     
       17. The method of claim 15, wherein the step of contacting the sample with an electrochemical sensor comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, and a sample chamber for holding the sample in electrolytic contact with the working electrode and counter electrode, wherein the sample chamber is sized to contain less than about 1 μL of sample and the sample chamber comprises a measurement zone bounded on at least two sides by the working electrode and the counter electrode. 
     
     
       18. The method of claim 15, wherein the step of contacting the sample with an electrochemical sensor comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, and a sample chamber for holding the sample in electrolytic contact with the working electrode and counter electrode, wherein the sample chamber is sized to contain less than about 1 μL of sample and the sample chamber comprises a measurement zone bounded on at least two sides by the working electrode and the counter electrode, the measurement zone having about a same volume as the sample chamber. 
     
     
       19. The method of claim 15, wherein the step of contacting the sample with an electrochemical sensor comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode disposed facing the working electrode, and a sample chamber for holding the sample in electrolytic contact with the working electrode and counter electrode, wherein the sample chamber is sized to contain less than about 1 μL of sample and the sample chamber comprises a measurement zone between the working electrode and the counter electrode. 
     
     
       20. A method of determining a concentration of an analyte in a sample, the method comprising steps of: contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, and a sample chamber for holding the sample in electrolytic contact with the working electrode and counter electrode;   holding the sample within the sample chamber in a non-flowing manner;   applying a potential between the working and counter electrodes to electrolyze the analyte in a portion of the sample within the sample chamber, wherein the portion of the sample in which the analyte is electrolyzed has a volume of less than 1 μL;   measuring current generated by the electrochemical sensor at a plurality of times while the analyte in the portion of the sample is being electrolyzed; and   determining, by coulometry, a concentration of the analyte in the sample using the measured currents.   
     
     
       21. The method of claim 20, wherein the step of applying a potential comprises applying a potential between the working and counter electrodes to electrolyze the analyte in a portion of the sample within the sample chamber, wherein the portion of the sample in which the analyte is electrolyzed has a volume of less than 0.5 μL. 
     
     
       22. The method of claim 20, wherein the step of applying a potential comprises applying a potential between the working and counter electrodes to electrolyze the analyte in a portion of the sample within the sample chamber, wherein the portion of the sample in which the analyte is electrolyzed has a volume of less than 0.2 μL. 
     
     
       23. The method of claim 20, wherein the step of contacting the sample with an electrochemical sensor comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, a sample chamber for holding the sample in electrolytic contact with the working electrode and counter electrode, and redox mediator disposed in the sample chamber. 
     
     
       24. The method of claim 20, wherein the step of contacting the sample with an electrochemical sensor comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, a sample chamber for holding the sample in electrolytic contact with the working electrode and counter electrode, and an electron transfer agent disposed in the sample chamber. 
     
     
       25. The method of claim 20, wherein the step of determining a concentration of the analyte in the sample comprises determining, by coulometry, a concentration of glucose in the sample using the measured currents. 
     
     
       26. The method of claim 20, wherein the step of determining a concentration of the analyte in the sample comprises determining, by coulometry, a concentration of glucose in a sample of blood using the measured currents. 
     
     
       27. The method of claim 20, wherein the step of applying a potential comprises electrolyzing the analyte in the portion of the sample for a period of no more than one minute. 
     
     
       28. The method of claim 20, wherein the step of contacting the sample comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode facing the working electrode, and a sample chamber for holding the sample in electrolytic contact with the working electrode and counter electrode. 
     
     
       29. The method of claim 20, wherein the step of contacting the sample comprises contacting the sample with an electrochemical sensor comprising a substrate, a working electrode disposed on the substrate, a counter electrode disposed on the substrate with the working electrode, and a sample chamber for holding the sample in electrolytic contact with the working electrode and counter electrode. 
     
     
       30. The method of claim 20, wherein the step of contacting the sample comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, and a sample chamber for holding the sample in electrolytic contact with the working electrode and counter electrode, wherein the electrochemical sensor is configured and arranged to generate a current, by electrolysis of a buffer solution with 10 mM glucose in the sample chamber, that is at least nine times greater than a current generated when a buffer solution with no glucose is in the sample chamber. 
     
     
       31. The method of claim 20, wherein the step of contacting the sample comprises contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, and a sample chamber for holding the sample in electrolytic contact with the working electrode and counter electrode, wherein the electrochemical sensor is configured and arranged to generate a current, by electrolysis of a buffer solution with 3 mM glucose in the sample chamber, that is at least 1.8 times greater than a current generated when a buffer solution with no glucose is in the sample chamber. 
     
     
       32. A method for determining a concentration of an analyte in a sample, comprising the steps of: contacting a sample with an electrochemical sensor comprising: a facing electrode pair comprising a working electrode and a counter electrode; and   a sample chamber for holding the sample in electrolytic contact with the working electrode, the sample chamber comprising a measurement zone positioned between the working and counter electrodes, wherein the measurement zone is sized to contain a volume of less than about 1 μL of sample;   holding the sample stationary in the sample chamber; and   determining the concentration of the analyte in the sample by coulometry.     
     
     
       33. The method of claim 32, wherein the measurement zone is sized to contain a volume of less than about 0.5 μL of sample. 
     
     
       34. The method of claim 33, wherein the measurement zone is sized to contain a volume of less than about 0.2 μL of sample. 
     
     
       35. The method of claim 34, wherein the measurement zone is sized to contain a volume of less than about 0.1 μL of sample. 
     
     
       36. The method of claim 32, wherein the sample chamber is sized to contain a volume of less than about 1 μL of sample. 
     
     
       37. The method of claim 36, wherein the sample chamber is sized to contain a volume of less than about 0.5 μL of sample. 
     
     
       38. The method of claim 37, wherein the sample chamber is sized to contain a volume of less than about 0.2 μL of sample. 
     
     
       39. The method of claim 32, wherein the step of determining the concentration of the analyte comprises: electrolyzing at least 90% of analyte present in the measurement zone by applying a potential across the working and counter electrodes;   determining an electrical charge used to electrolyze the analyte; and   correlating the electrical charge with the concentration of the analyte in the sample.   
     
     
       40. The method of claim 39, wherein at least 90% of the analyte is electrolyzed in less than about 5 minutes. 
     
     
       41. The method of claim 40, wherein at least 90% of the analyte is electrolyzed in less than about 1 minute. 
     
     
       42. The method of claim 39, wherein the step of determining an electrical charge comprises the steps of: measuring a current generated at the working electrode at two or more times as the analyte is electrolyzed; and   integrating the measured currents over time to obtain the electrical charge used to electrolyze the analyte.   
     
     
       43. The method of claim 32, wherein the step of determining the concentration of the analyte by coulometry comprises the steps of: electrolyzing a portion of the analyte by applying a potential across the working and the counter electrode;   measuring a current generated at the working electrode at two or more times during the electrolysis;   extrapolating a current curve based on the measured currents;   integrating the current curve over time to obtain an electrical charge necessary to electrolyze at least 90% of the analyte; and   correlating the electrical charge with the concentration of the analyte in the sample.   
     
     
       44. The method of claim 32, wherein the sensor further comprises a non-leachable enzyme on the working electrode. 
     
     
       45. The method of claim 44, wherein the enzyme is immobilized on the working electrode. 
     
     
       46. The method of claim 32, wherein the sensor further comprises a non-leachable redox mediator on the working electrode. 
     
     
       47. The method of claim 46, wherein the redox mediator is immobilized on the working electrode. 
     
     
       48. The method of claim 46, wherein the redox mediator comprises a polymer and a redox species ionically, covalently, or coordinatively bound to the polymer. 
     
     
       49. The method of claim 48, wherein the redox species is coordinatively bound to the polymer. 
     
     
       50. The method of claim 46, wherein the redox mediator comprises an air-oxidizable redox mediator. 
     
     
       51. The method of claim 50, wherein the air-oxidizable redox mediator comprises Os[4,4'-dimethoxy-2,2'-bipyridine] 2  Cl +/+2  or Os[4,7-dimethoxy-1,10-phenanthroline] 2  Cl +/+2  complexed with poly(1-vinyl imidazole) or poly(4-vinylpyridine). 
     
     
       52. The method of claim 50, wherein the redox mediator comprises a transition metal complex. 
     
     
       53. The method of claim 52, wherein the transition metal complex comprises an osmium complex. 
     
     
       54. The method of claim 53, wherein the osmium complex comprises osmium complexed with at least one ligand having a nitrogen-containing heterocycle. 
     
     
       55. The method of claim 54, wherein the ligand having a nitrogen-containing heterocycle comprises 2,2'-bipyridine, 1,10-phenanthroline, or a derivative thereof. 
     
     
       56. The method of claim 55, wherein the ligand having a nitrogen-containing heterocycle comprises a mono-, di-, or polyalkoxy derivative of 2,2'-bipyridine or 1,10-phenanthroline, wherein the carbon to oxygen ratio of the alkoxy functional groups is sufficient to retain solubility of the transition metal complex in water prior to crosslinking. 
     
     
       57. The method of claim 56, wherein the nitrogen-containing heterocycle comprises 4,4'-dialkoxy-2,2 '-bipyridine-or 4,7-dialkoxy-1,10-phenanthroline, wherein the carbon to oxygen ratio of the alkoxy functional groups is sufficient to retain solubility of the transition metal complex in water prior to crosslinking. 
     
     
       58. The method of claim 57, wherein the nitrogen-containing heterocycle comprises 4,4'-dimethoxy-2,2 '-bipyridine or 4,7-dimethoxy-1,10-phenanthroline. 
     
     
       59. The method of claim 53, wherein the osmium complex comprises osmium complexed with a polymeric ligand. 
     
     
       60. The method of claim 59, wherein the polymeric ligand comprises a nitrogen-containing heterocycle. 
     
     
       61. The method of claim 60, wherein the polymer comprises poly(4-vinyl pyridine) or poly (1-vinyl imidazole). 
     
     
       62. The method of claim 46, wherein at least 90% of the redox mediator is in an oxidized state prior to introduction of the sample in the sensor. 
     
     
       63. The method of claim 46, wherein the sensor further comprises a non-leachable second electron transfer agent on the working electrode. 
     
     
       64. The method of claim 63, wherein the second electron transfer agent is immobilized on the working electrode. 
     
     
       65. The method of claim 63, wherein the second electron transfer agent comprises an enzyme. 
     
     
       66. The method of claim 65, wherein the analyte is glucose. 
     
     
       67. The method of claim 66, wherein the enzyme is a glucose oxidase. 
     
     
       68. The method of claim 32, wherein the sensor further comprises sorbent material disposed in the measurement zone. 
     
     
       69. The method of claim 68, wherein the step of contacting the sample with an electrochemical sensor further comprises contacting the sample with the sorbent material to wick the sample into the measurement zone. 
     
     
       70. The method of claim 62, wherein the working electrode and counter electrode have a separation distance of less than about 0.2 mm. 
     
     
       71. The method of claim 70, wherein the separation distance is less than about 0.1 mm. 
     
     
       72. The method of claim 71, wherein the separation distance is less than about 0.05 mm. 
     
     
       73. The method of claim 62, wherein the sensor comprises two or more facing electrode pairs. 
     
     
       74. The method of claim 32, wherein the sample chamber and the measurement zone have a same size. 
     
     
       75. The method of claim 32, further comprising transporting the sample into the sample chamber prior to holding the sample within the sample chamber. 
     
     
       76. The method of claim 32, wherein holding the sample within the sample chamber in a non-flowing manner comprises transporting fluid through the sample chamber, and   halting the transport of the fluid through the sample chamber to provide a sample within the sample chamber, the transport of the fluid being halted at least until after the concentration of the analyte in the sample is determined by coulometry.   
     
     
       77. The method of claim 32, wherein holding the sample within the sample chamber in a non-flowing manner comprises transporting the sample into the sample chamber, and   halting the transport of sample into the sample chamber when the sample chamber is substantially full.   
     
     
       78. A method of determining the concentration of an analyte in a sample, comprising the steps of: contacting a sample with an electrochemical sensor, wherein the sensor comprises a working electrode, a non-leachable redox mediator on the working electrode, and a sample chamber sized to contain a volume of less than about 1 μL of sample in electrolytic contact with the working electrode;   holding the sample stationary in the sample chamber; and   determining the concentration of the analyte in the sample by coulometry.   
     
     
       79. The method of claim 78, wherein the sample chamber is sized to contain a volume of less than about 0.5 μL of sample. 
     
     
       80. The method of claim 79, wherein the sample chamber is sized to contain a volume of less than about 0.2 μL of sample. 
     
     
       81. The method of claim 78, wherein the redox mediator is immobilized on the working electrode. 
     
     
       82. The method of claim 78, wherein the sensor further comprises a non-leachable second electron transfer agent on the working electrode. 
     
     
       83. The method of claim 82, wherein the second electron transfer agent is immobilized on the working electrode. 
     
     
       84. The method of claim 82, wherein the second electron transfer agent comprises an enzyme. 
     
     
       85. The method of claim 78, wherein the sensor further comprises a sorbent material within the sample chamber to reduce the volume of sample that the sample chamber is sized to hold. 
     
     
       86. The method of claim 78, wherein the redox mediator comprises a transition metal complex. 
     
     
       87. The method of claim 86, wherein the transition metal complex is an osmium, ruthenium, iron, or cobalt complex. 
     
     
       88. The method of claim 87, wherein the transition metal complex comprises an osmium complex. 
     
     
       89. The method of claim 88, wherein the osmium complex comprises osmium complexed with at least one ligand having a nitrogen-containing heterocycle. 
     
     
       90. The method of claim 89, wherein the ligand having a nitrogen-containing heterocycle comprises 2,2'-bipyridine, 1,10-phenanthroline, or a derivative thereof. 
     
     
       91. The method of claim 90, wherein the ligand having a nitrogen-containing heterocycle comprises a mono-, di-, or polyalkoxy derivative of 2,2'-bipyridine or 1,10-phenanthroline, wherein the carbon to oxygen ratio of the alkoxy functional groups is sufficient to retain solubility of the transition metal complex in water prior to crosslinking. 
     
     
       92. The method of claim 90, wherein the nitrogen-containing heterocycle comprises 2,2'-bipyridine, 4,4'-dimethyl-2,2'-bipyridine, 4,4'-dialkoxy-2,2'-bipyridine, 1,10-phenanthroline, 4,7-dimethyl-1,10-phenanthroline, or 4,7-dialkoxy-1,10-phenanthroline, wherein the carbon to oxygen ratio of the alkoxy functional groups is sufficient to retain solubility of the transition metal complex in water prior to crosslinking. 
     
     
       93. The method of claim 88, wherein the osmium complex comprises osmium complexed with a polymeric ligand. 
     
     
       94. The method of claim 93, wherein the polymeric ligand comprises a nitrogen-containing heterocycle. 
     
     
       95. The method of claim 94, wherein the polymer comprises poly(4-vinyl pyridine) or poly(1-vinyl imidazole). 
     
     
       96. A method for determining the concentration of an analyte in a sample, comprising the steps of: contacting the sample with an electrochemical sensor; the sensor comprising: an electrode pair comprising a working electrode and a counter electrode;   a sample chamber for holding the sample in electrolytic contact with the working electrode; and   a sorbent disposed within the sample chamber;   wherein the sample chamber is sized to hold less than about 1 μL of sample;   holding the sample stationary in the sample chamber; and   determining the concentration of the analyte by coulometry.     
     
     
       97. The method of claim 96, wherein the method further comprises wicking the sample into the sample chamber using the sorbent material. 
     
     
       98. The method of claim 96, wherein the electrode pair is a facing electrode pair. 
     
     
       99. A method for measuring analyte in a patient sample, the method comprising: contacting the patient with an analyte measuring device, the device comprising: sample acquisition means for producing a patient sample; and   an electrochemical sensor for measuring analyte in the sample; wherein the electrochemical sensor comprises: a facing electrode pair, comprising a working electrode and a counter electrode; and   a measurement zone positioned between the working electrode and counter electrode, wherein the measurement zone is sized to contain a volume of less than about 1 μL of sample;       acquiring a sample using the sample acquisition means;   transporting a portion of the sample to the measurement zone of the electrochemical sensor;   holding the sample stationary in the sample chamber; and   determining the concentration of the analyte in the sample by coulometry.   
     
     
       100. The method of claim 99, wherein the sample acquisition means comprises a skin-piercing member and the step of acquiring a sample comprises piercing the patient's skin to produce a sample. 
     
     
       101. The method of claim 100, wherein the skin piercing member comprises a lancet. 
     
     
       102. The method of claim 99, wherein the electrochemical sensor further comprises a sorbent material disposed within the measurement zone of the sensor. 
     
     
       103. The method of claim 102, wherein said transporting comprises: wicking the sample into the measurement zone using the sorbent material.   
     
     
       104. The method of claim 99, wherein the measurement zone is sized to contain a volume of less than about 0.5 μL of sample. 
     
     
       105. The method of claim 104, wherein the measurement zone is sized to contain a volume of less than about 0.2 μL of sample. 
     
     
       106. The sensor of claim 105, wherein the measurement zone is sized to contain a volume of less than about 0.1 μL of sample. 
     
     
       107. The method of claim 99, wherein the sensor further comprises a non-leachable redox mediator. 
     
     
       108. The method of claim 107, wherein the redox mediator is an air-oxidizable redox mediator. 
     
     
       109. A method for measuring analyte in a patient sample, the method comprising: contacting the patient with an analyte measuring device, the device comprising: sample acquisition means for producing a patient sample; and   an electrochemical sensor for measuring analyte in the sample, wherein the electrochemical sensor comprises: a working electrode;   non-leachable redox mediator on the working electrode; and   a sample chamber for holding the sample in electrolytic contact with the working electrode, wherein the sample chamber is sized to contain a volume of less than about 1 μL of sample;       acquiring a sample using the sample acquisition means;   transporting a portion of the sample to the sample chamber of the electrochemical sensor;   holding the sample within the sample chamber in a non-flowing manner; and   determining the concentration of the analyte in the sample by coulometry.   
     
     
       110. A method for determination of a concentration of an analyte in a sample, the method comprising the steps of: contacting a sample with an electrochemical sensor comprising: first and second electrode pairs, each pair comprising a working electrode, and a sample chamber for holding the sample in electrolytic contact with the working electrode, the sample chamber sized to contain a volume of less about 1 μL of sample; wherein said first electrode pair includes a non-leachable redox mediator and a non-leachable enzyme on the working electrode; and wherein said second electrode pair includes non-leachable redox mediator on the working electrode in the absence of enzyme;     holding the sample stationary in the sample chamber;   measuring substantially simultaneously, and at two or more times, a first current generated at the first electrode pair and a second current generated at the second electrode pair; and   integrating the measured first currents over time to obtain a first charge;   integrating the measured second currents over time to obtain a second charge;   subtracting the second charge from the first charge to obtain a noise-reduced charge; and   correlating the concentration of the analyte to the noise-reduced charge.   
     
     
       111. The method of claim 110, wherein the first and second electrode pairs are facing electrode pairs and further comprise a counter electrode and a measurement zone between the working electrode and counter electrode, the measurement zone sized to contain a volume of less than about 1 μL of sample. 
     
     
       112. A method for determining a concentration of an analyte in a sample, the method comprising the steps of: providing an electrochemical sensor, the sensor having one or more facing electrode pairs, each electrode pair comprising a working electrode, a counter electrode, and a measurement zone between the working electrode and the counter electrode, the measurement zone having a volume of less than about 1 μL, wherein the measurement zones of the one or more electrode pairs have approximately equal volumes, and wherein at least one of the electrode pairs further comprises redox mediator on its working electrode;   measuring a capacitance of at least one of the electrode pairs;   calculating the volume of the measurement zone from the capacitance measurement;   contacting the sample with the sensor; and   determining the concentration of the analyte in the sample by coulometry.   
     
     
       113. The method of claim 112, wherein the redox mediator is a non-leachable redox mediator. 
     
     
       114. A method for determining a concentration of an analyte in a sample comprising the steps of: contacting the sample with an electrochemical sensor, the sensor comprising a working electrode and a non-leachable redox mediator on the working electrode, wherein the molar amount of redox mediator in a reduced form prior to introduction of the sample is less than, on a stoichiometric basis, 5% of the expected molar amount of the analyte to be electrolyzed;   holding the sample stationary in the sample chamber;   electrolyzing less than about 1 μL of sample; and   determining the concentration of the analyte in the sample by coulometry.   
     
     
       115. A method of determining a concentration of an analyte in a sample comprising the steps of: contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, and a measurement zone bounded on at least two sides by the working and the counter electrodes, wherein the measurement zone is sized to contain less than about 1 μL of sample;   holding the sample stationary in the sample chamber; and   determining the concentration of the analyte in the sample by coulometry.